Biomimetic peptide and biodegradable delivery platform for the treatment of angiogenesis- and lymphangiogenesis-dependent diseases

ABSTRACT

Mimetic peptides having anti-angiogenic and anti-tumorigenic properties and methods of their use for treating cancer, ocular diseases, such as age-related macular degeneration, and other-angiogenesis-dependent diseases are disclosed. More particularly, an isolated peptide comprising the amino acid sequence LRRFSTAPFAFIDINDVINF, which exhibits anti-angiogenic activity in endothelial cell proliferation, migration, adhesion, and tube formation assays, anti-migratory activity in human breast cancer cells in vitro, anti-angiogenic and anti-tumorigenic activity in vivo in breast cancer xenograft models, and age-related macular degeneration models is disclosed. The isolate peptide also exhibits anti-lymphangiogenic and directly anti-tumorigenic properties.

This application is a continuation of U.S. patent application Ser. No.14/896,521, filed Dec. 7, 2015 and issued as U.S. Pat. No. 9,802,984 onOct. 31, 2017. U.S. Ser. No. 14/896,521 claims the benefit of U.S.Provisional Application No. 61/832,290, filed Jun. 7, 2013. The contentsof the aforementioned patent applications are incorporated herein byreference in their entireties.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R21 CA131931 andR01 CA138264 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“ASX-006C1_Sequence_Listing_ST25.txt”. The sequence listing is 2,381bytes in size, and was electronically filed via EFS-Web on Sep. 22,2017. The sequence listing is hereby incorporated by reference in itsentirety.

BACKGROUND

Cancer is a major public health problem in the United States and otherparts of the world. Currently, 1 in 4 deaths in the United States is dueto cancer. Angiogenesis plays a critical role in tumor growth andmetastasis in most types of cancer. In particular, its importance hasbeen demonstrated in breast cancer, the most commonly diagnosed femalemalignancy in the United States. Anti-angiogenic therapeutics, either asa monotherapy or in combination with other therapeutics, are promisingand are being intensely investigated in both preclinical and clinicalstudies. Anti-VEGF therapeutics showed early promise in clinical trials;however, although an anti-VEGF antibody bevacizumab (Genentech/Roche)was approved by the Food and Drug Administration (FDA) for breast cancerin 2008 in combination with chemotherapy, in November 2011, the FDArevoked the breast cancer indication because it has not demonstrated anoverall survival benefit.

The development of anti-angiogenic therapies to treat breast and othercancers, as well as, ocular proliferative diseases, such as age-relatedmacular degeneration, is ongoing. Lymphangiogenesis also plays animportant role in cancer metastasis (Holopainen et al., 2011). To date,no peptide drugs have been approved for the treatment of cancer or otherangiogenesis- and lymphangiogenesis-dependent diseases.

Peptides have been employed as therapeutics for multiple diseases andrecently have been investigated in clinical applications to targettumors either for imaging or therapy (Folkman, 2010; Senger et al.,1983; Leung et al., 1989; Carmeliet, 2005; Carmeliet and Jain, 2000;Carmeliet and Jain, 2011; Rosca et al., 2011). Mimetic peptides arepeptides that biologically mimic active determinants on biomolecules. Ingeneral, peptides are attractive tools as therapeutics due to theirspecific target binding, ability to penetrate cells and ease ofmodification giving flexibility for different applications (Carmelietand Jain, 2000; Folkman, 2006). In addition, they are less toxic becausethey bind to their targets with high specificity and they areinexpensive to produce.

Some of the properties that make peptides attractive candidates,however, also contribute to their disadvantages. Although peptides caninteract specifically with cellular receptors, sometimes theseinteractions may be of low affinity. In addition, the use of peptides astherapeutic agents is currently limited due to their short half-life andreduced bioavailability. Attempts to modify a peptide to increase itsbioavailability include substitution with non-natural amino acids,pegylation of the peptide, and delivery of the peptide in a nano- ormicro-particle.

SUMMARY

The presently disclosed subject matter provides peptide compositions,methods, and kits for treating a disease, disorder, or dysfunction thatis related to angiogenesis, lymphangiogenesis, vascular permeabilityand/or tumorigenesis. The presently disclosed peptide compositions andmethods, in some aspects, inhibit angiogenesis, lymphangiogenesis,vascular permeability and/or tumorigenesis, which play a critical rolein multiple diseases or disorders. Accordingly, in some aspects, thecompositions and methods of the presently disclosed subject matter allowthe prevention or reduction of blood vessel, lymphatic vessel, or tumorformation involving a cell, tissue or organ.

In some aspects, the presently disclosed subject matter provides anisolated peptide comprising an amino acid sequence at least 85%identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO:1), wherein the peptideexhibits anti-angiogenic, anti-vascular permeability, anti-tumorigenesisand/or anti-lymphangiogenic properties.

In other aspects, the presently disclosed subject matter provides acomposition comprising a pharmaceutically acceptable carrier and aneffective amount of an isolated peptide comprising an amino acidsequence at least 85% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO:1),wherein the peptide exhibits anti-angiogenic, anti-vascularpermeability, anti-tumorigenesis and/or anti-lymphangiogenic properties.

In further aspects, the presently disclosed subject matter provides akit comprising an isolated peptide comprising an amino acid sequence atleast 85% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO:1), wherein thepeptide exhibits anti-angiogenic, anti-vascular permeability,anti-tumorigenesis and/or anti-lymphangiogenic properties.

In still further aspects, the presently disclosed subject matterprovides a nanoparticle or microparticle comprising an isolated peptidecomprising an amino acid sequence at least 85% identical toLRRFSTAPFAFIDINDVINF (SEQ ID NO:1), wherein the peptide exhibitsanti-angiogenic, anti-vascular permeability, anti-tumorigenesis and/oranti-lymphangiogenic properties.

In some aspects, the presently disclosed subject matter provides amethod for inhibiting angiogenesis, lymphangiogenesis, vascularpermeability and/or tumorigenesis involving a cell, the methodcomprising: contacting the cell with an isolated peptide comprising anamino acid sequence at least 85% identical to LRRFSTAPFAFIDINDVINF (SEQID NO:1), in an amount sufficient to inhibit angiogenesis,lymphangiogenesis, vascular permeability and/or tumorigenesis involvingthe cell.

In some other aspects, the presently disclosed subject matter provides amethod for treating a subject suffering from a disease related toangiogenesis, lymphangiogenesis, vascular permeability, and/ortumorigenesis or to prevent or delay a subject from developing a diseaserelated to angiogenesis, lymphangiogenesis, vascular permeability,and/or tumorigenesis, the method comprising: administering to thesubject an isolated peptide comprising an amino acid sequence at least85% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO:1), in an amountsufficient to treat, delay, or prevent the disease in the subject.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIGS. 1A-1C show proliferation (Panel A), adhesion (Panel B), andmigration (Panel C) using the SP2043 peptide (SEQ ID NO:1) with humanumbilical vein endothelial cells (HUVEC);

FIGS. 2A-2C show adhesion (Panel A), proliferation (Panel B), andmigration (Panel C) using the SP2043 peptide (SEQ ID NO:1) with humanretinal endothelial cells (HREC);

FIGS. 3A-3D show a HUVEC tube formation assay using a CM control (PanelA) and the SP2043 peptide (SEQ ID NO:1) (25 μM SP2043 peptide; Panel B);and a microvascular endothelial cell (MEC) tube formation assay using acontrol (Ctrl; Panel C) and the SP2043 peptide (SEQ ID NO:1) (25 μMSP2043 peptide; Panel D);

FIG. 4 shows the effect of the SP2043 peptide (SEQ ID NO:1), alsoreferred to herein and in the Figures as SEQ.1, on lymphatic endothelialcell (LEC) adhesion;

FIG. 5 shows the effect of the SP2043 peptide (SEQ ID NO:1) on lymphaticendothelial cell (LEC) tube formation;

FIGS. 6A-6B shows the effect of the SP2043 peptide (SEQ ID NO:1) onhepatocyte growth factor (HGF) and insulin growth factor 1 (IGF1)signaling in vitro with microvascular endothelial cells (MEC; Panel A)and lymphatic endothelial cells (LEC; Panel B);

FIG. 7 shows the effect of the SP2043 peptide (SEQ ID NO:1) on theproliferation of triple-negative breast cancer cell lines MDA-MB-231 andSUM149, and the estrogen receptor positive cell line MCF-7;

FIG. 8 shows the effect of the SP2043 peptide (SEQ ID NO:1) onhepatocyte growth factor (HGF) signaling in MDA-MB-231 cells;

FIG. 9 shows the effect of the SP2043 peptide (SEQ ID NO:1) on thegrowth of MDA-MB-231 orthotopic tumors in SCID mice over 33 days (ip,intraperitoneal injection);

FIGS. 10A-10B show the effect of the SP2043 peptide (SEQ ID NO:1) (PanelB) on phosphorylation of c-Met and angiogenesis in vivo (Panel A,control);

FIGS. 11A-11G show the effect of the SP2043 peptide (SEQ ID NO:1) onangiogenesis as seen by lectin staining for immunohistochemistry (IHC)and LYVE-1 staining for lymphangiogenesis in vivo: control stained withlectin (Panel A) and LYVE-1 (Panel B); the SP2043 peptide (SEQ ID NO:1)stained with lectin (Panel C) and LYVE-1 (Panel D); control and theSP2043 peptide (SEQ ID NO:1) (Panel E); and pixel density using lectin(Panel F) and LYVE-1 (Panel G);

FIGS. 12A-12B show the effect of the SP2043 peptide (SEQ ID NO:1) on themetastasis of MDA-MB-231-luc tumors in multiple organs in thetumor-conditioned media pre-treated metastasis model by photon flux fromtumor cells (Panel A) and the effect of the SP2043 peptide (SEQ ID NO:1)on the metastasis of MDA-MB-231-luc tumors to lymph nodes as seen bystaining with vimentin antibody for the presence of human cells (PanelB);

FIGS. 13A-13E show the effect of the SP2043 peptide (SEQ ID NO:1) onneovascularization as compared to aflibercept in a CNV model (PanelsA-B) and as compared to a control in a rho-VEGF mouse model (Panel C-E);

FIG. 14 shows the effect of the SP2043 peptide (SEQ ID NO:1) on theneovasculature in a laser induced CNV model in the mouse eye;

FIGS. 15A-15B show the effect of the SP2043 peptide (SEQ ID NO:1) onvascular leakage in the Tet/Opsin/VEGF mouse model;

FIG. 16 shows the effect of the SP2043 peptide (SEQ ID NO:1) on VEGFmediated vascular permeability in the rabbit eye;

FIG. 17 shows a representative example of SP2043 peptide (SEQ ID NO:1)loaded (2%) in poly(lactic-co-glycolic acid) (PLGA) microparticles; PLGAwas used in a L:G ratio of 65/35, MW=40-75 kDa. Scale bar is 10 microns;

FIGS. 18A-18B show: (Panel A) 2% and 5% SP2043 peptide (SEQ ID NO:1)loaded PLGA microparticles and sizing; Number averaged particledistribution (Num. Avg) and volume averaged particle distribution (Vol.Avg) are indicated (Mean+SD); and (Panel B) the zeta potentials (surfacecharge) of 2% and 5% SP2043 peptide (SEQ ID NO:1) loaded microparticles;

FIG. 19A and FIG. 19B show the controlled release of a labeled peptideanalog of SP2043 from PLGA microparticles under physiological conditionsin situ;

FIG. 20 shows the controlled release of the SP2043 peptide (SEQ ID NO:1)without any label or modification in PLGA microparticles in situ underphysiological conditions (method shown in FIG. 32 below);

FIG. 21 shows SEM imaging of PLGA microparticles incorporating 2% byweight SP2043 (left) and 5% by weight 2043 (right). PLGA in this exampleused L:G ratio of 85/15, MW=190-240 kDa. Scale bar is 10 microns;

FIG. 22 shows the SP2043 peptide (SEQ ID NO:1) encapsulated intonanoparticles PLGA 65/35 was used. Ellipsoidal nanoparticles (left) andspherical nanoparticles (right);

FIG. 23 shows the effect of the PLGA 85/15 microparticle encapsulatingthe SP2043 peptide (SEQ ID NO:1) on regression in mouse eyes followinglaser-induced choroidal neovascularization in a mouse model (2 week datashown);

FIG. 24 shows the effect of PLGA 85/15 microparticles containing theSP2043 peptide (SEQ ID NO:1) on neovascularization overtime in alaser-induced wet AMD mouse model;

FIG. 25 shows the effect of PLGA 65/35 microparticles containing theSP2043 peptide (SEQ ID NO:1) on neovascularization overtime in alaser-induced wet AMD mouse model;

FIG. 26 shows the effect of the SP2043 peptide (SEQ ID NO:1) on theinhibition of neovascularization in a rabbit model. Data at day 34following intravitreal injection into rabbit eyes;

FIG. 27 shows PLGA (85/15) encapsulating the SP2043 peptide (SEQ IDNO:1) in rho/VEGF transgenic mice;

FIG. 28 shows the effect of the SP2043 peptide (SEQ ID NO:1) incombination with other anti-angiogenesis agents in a laser-inducedchoroidal neovascularization model in mice;

FIG. 29 shows the effect of polymers, such as PBAEs, on the ability ofthe SP2043 peptide (SEQ ID NO:1) to self-assemble into approximately100-nm nanoparticles. Polymer 447 refers to(3-aminopropyl)-4-methylpiperazine end-capped poly(1,4-butanedioldiacrylate-co-4-amino-1-butanol) and polymer 657 refers to(3-aminopropyl)-4-methylpiperazine end-capped poly(1,6-hexanedioldiacrylate-co-5-amino-1-pentanol) (B6-S5-E7). +/− refers to with orwithout the SP2043 peptide (SEQ ID NO:1). Particle size is larger andnanoparticle concentration larger when the SP2043 peptide (SEQ ID NO:1)is together with polymer for self-assembly;

FIG. 30 shows PLGA and PLGA-PEG nanoparticles encapsulating SP2043;

FIGS. 31A-31B show the effect of systemically intravenously injectedSP2043 peptide (SEQ ID NO:1) containing nanoparticles or free peptide onaccumulation in tumor, as well as other organs. FIG. 31A showsaccumulation per organ and FIG. 31B shows total accumulation. In bothfigures, the term “naked” refers to the free peptide, while the terms“sphere,” “ellipse,” and “PEG,” i.e., polyethylene glycol, refer tothree different types of nanoparticles containing the SP2043 peptide(SEQ ID NO:1); and

FIGS. 32A-32B show a method for quantifying the presently disclosedpeptides by using electrophoresis and staining with SimplyBlue (Panel A)followed by mass spectrometry (Panel B). Panel A contained the followingsamples: lane 1, marker; lane 2, 4 μg SP2043 peptide (SEQ ID NO:1)positive control; lane 3, 4 μg SP2043 peptide (SEQ ID NO:1) in PBSmedium; lane 4, 4 μg SP2043 peptide (SEQ ID NO:1) in FBS medium.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the presently disclosed subject matter areshown. Like numbers refer to like elements throughout. The presentlydisclosed subject matter may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements. Indeed, many modifications andother embodiments of the presently disclosed subject matter set forthherein will come to mind to one skilled in the art to which thepresently disclosed subject matter pertains having the benefit of theteachings presented in the foregoing descriptions and the associatedFigures. Therefore, it is to be understood that the presently disclosedsubject matter is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims.

I. Mimetic Peptides Derived from Collagen Type IV

Peptides generally offer many advantages over other types of therapiesfor certain diseases in that they are non-immunogenic, less toxicbecause they bind to their targets with high specificity, and areinexpensive to produce. See, e.g., International PCT Patent PublicationNo. WO 2008/085828 and International PCT Patent Application PublicationNo. WO 2007/033215, each which is incorporated herein by reference inits entirety. The presently disclosed peptides exhibit anti-angiogenic,anti-vascular permeability, anti-tumorigenesis and/oranti-lymphangiogenic properties, which could lead to an increase inoverall survival in certain diseases. For example, the presentlydisclosed peptides may benefit cancer patients and may help treat ocularproliferative diseases, such as age-related macular degeneration anddiabetic retinopathy.

In general, the presently disclosed peptides are characterized by havingthe motif comprising the amino acid sequence LRRFSTXPXXXXDINDVXNF (SEQID No: 2). Peptides characterized by having the motif comprising theamino acid sequence LRRFSTXPXXXXNINNVXNF (SEQ ID No: 4) have beendisclosed in International Publication WO 2012/079088 (incorporatedherein by reference in its entirety). This motif was determined bymaking substitutions in the pentastatin-1 peptide. The WO 2012/079088publication disclosed that, in some embodiments, the positions in SEQ IDNo: 3 denoted by X could be varied and the resulting peptide could stillbe used for, and in some embodiments, was better for, inhibitingangiogenesis, lymphangiogenesis, vascular permeability and/ortumorigenesis, and for treatment of a subject thereof while otherpositions in SEQ ID No: 3 could not be varied to retain inhibitorycharacteristics. In other embodiments, it was found that X at thefollowing positions could have the following substitutions in SEQ ID No:3: position 7 could be M, A, or G; position 9 could be F, A, Y, or G;position 10 could be M, A, G, dA, or Nle; position 11 could be F, A, Y,G, or 4-ClPhe; position 12 and position 18 could be Abu, G, S, A, V, T,I, L or AllyGly. In an embodiment, the peptide characterized by havingthe motif comprising the amino acid sequence LRRFSTAPFAFININNVINF (SEQID No: 3; also called SP2036) was disclosed.

The presently disclosed peptides are characterized by having the motifcomprising the amino acid sequence LRRFSTXPXXXXDINDVXNF (SEQ ID No:2),which differs from previously disclosed SEQ ID No: 4 at positions 13 and16 (Table 1). In a particular embodiment, a peptide characterized byhaving the motif comprising the amino acid sequence LRRFSTAPFAFIDINDVINF(SEQ ID No: 1; also called SP2043 and SEQ.1) is disclosed which differsfrom previously disclosed SEQ ID No: 3 (also called SP2036) at positions13 and 16 (Table 1). It is disclosed herein that the substitution ofaspartate at positions 13 and 16 results in a peptide that is lesshydrophobic, but still can be used for inhibiting angiogenesis,lymphangiogenesis, vascular permeability and/or tumorigenesis involvinga cell, tissue, or organ, and for treatment of a subject thereof.Further, because this peptide is less hydrophobic, it is easier toproduce.

TABLE 1 Representative Peptides SEQ ID NO: 1 LRRFST A P FAFI DINDV INFSP2043 SEQ ID NO: 2 LRRFST X P XXXX DINDV XNF SEQ ID NO: 3LRRFST A P FAFI NINNV INF SP2036 SEQ ID NO: 4 LRRFST X P XXXX NINNV XNF

A. Representative Embodiments

In a particular embodiment, the isolated peptide comprises the aminoacid sequence LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1).

In some embodiments, the peptide comprises the amino acid sequenceLRRFSTXPXXXXDINDVXNF (SEQ ID NO: 2), and wherein X is any amino acid. Xmay be a natural or non-natural amino acid.

In other embodiments, the peptide comprises the amino acid sequenceLRRFSTXPXXXXDINDVXNF (SEQ ID NO:2), wherein X at position 7 is M, A, orG; X at position 9 is F, A, Y, or G; X at position 10 is M, A, G, dA, orNle; X at position 11 is F, A, Y, G, or 4-ClPhe; X at position 12 andposition 18 are Abu, G, S, A, V, T, I, L or AllyGly. Each substitutionat an X residue in this embodiment has either been tested (as shown inInternational PCT Patent Application Publication No. WO 2012/079088,which is incorporated herein by reference in its entirety, or in theExamples provided herein below) or is a conservative amino acidsubstitution for a tested amino acid.

In some embodiments, the presently disclosed peptides have several Xresidues that may be any amino acid, whether natural or non-natural (X7,X9, X10, X11, X12, and X18). By natural amino acids, it is meant thoseamino acids that occur in nature, such as glycine, alanine, valine,leucine, isoleucine, serine, threonine, aspartic acid, asparagine,lysine, glutamic acid, glutamine, arginine, histidine, phenylalanine,cysteine, tryptophan, tyrosine, methionine, proline, pyrrolysine, andselenocysteine. By non-natural amino acids, it is meant amino acids thatdo not occur in nature, but that can be incorporated into a polypeptidechain. Non-natural amino acids include, but are not limited to2-aminobutyric acid (Abu), norleucine (Nle), 4-chloro phenylalanine(4-ClPhe), allylglycine (AllyGly) and other non-natural amino acids suchas those detailed in Ma (2003). Amino acid analogs that are known in theart may be employed in the presently disclosed subject matter.

A “peptide” or “protein” comprises a string of at least three aminoacids linked together by peptide bonds. The terms “protein” and“peptide” may be used interchangeably. Peptide may refer to anindividual peptide or a collection of peptides. Also, one or more of theamino acids in a presently disclosed peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, and the like. In some embodiments, the modifications ofthe peptide lead to a more stable peptide (e.g., greater half-life invivo). In other embodiments, other modifications may include cyclizationof the peptide, the incorporation of D-amino acids, other moleculesconjugated to the N-terminus and C-terminus, conjugation of fluorescentprobes, biomolecules, such as poly(ethylene glycol), targeting ligands,and the like, retro-inversion and the like. None of the modificationsshould substantially interfere with the desired biological activity ofthe peptide.

By “Collagen IV derived peptide” it is meant a peptide comprising aC-N-X(3)-V-C or P-F-X(2)-C or LX(2)FX(3)PFX(2)CNX(4)CNX collagen motif.If desired, the peptide includes at least about 5, 10, 20, 30, 40, 50 ormore amino acids that flank the carboxy or amino terminus of the motifin the naturally occurring amino acid sequence. Type IV collagen derivedpeptides include, for example, pentastatin-1, tumstatin, and targetingRGD. By “alteration” is meant a change in the sequence or in amodification (e.g., a post-translational modification) of a gene orpolypeptide relative to an endogeneous wild-type reference sequence.

By an “isolated peptide” is meant a presently disclosed peptide that hasbeen separated from components that naturally accompany it. Typically,the peptide is isolated when it is at least 60%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight, apresently disclosed peptide. An isolated peptide may be obtained, forexample, by extraction from a natural source, by expression of arecombinant nucleic acid encoding such a peptide; or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, for example, column chromatography, polyacrylamide gelelectrophoresis, or by HPLC analysis.

By “substantially identical” is meant a peptide, a polypeptide ornucleic acid molecule exhibiting at least 50% identity to a referenceamino acid sequence (for example, any one of the amino acid sequencesdescribed herein) or nucleic acid sequence. Preferably, such a sequenceis at least 60%, more preferably 80% or 85%, and even more preferably90%, 95% or even 99% identical at the amino acid level or nucleic acidto the sequence used for comparison.

“Functional variants” of SEQ ID NO:1 and SEQ ID NO:2 include functionalfragments and/or functional fusion peptides. A functional variant of thepresently disclosed sequences refers to an isolated and/or recombinantpeptide which has at least one property, activity and/or functioncharacteristic of peptide encoded by SEQ ID NO:1 or SEQ ID NO:2, such asexhibiting anti-angiogenic, anti-vascular permeability,anti-tumorigenesis and/or anti-lymphangiogenic properties, which couldlead to an increase in overall survival in certain diseases. Generally,fragments or portions of the presently disclosed peptides encompassed bythe presently disclosed subject matter include those having a deletion(i.e., one or more deletions) of an amino acid (i.e., one or more aminoacids) relative to the peptides encoded by SEQ ID NO: 1 or SEQ ID NO: 2(such as N-terminal, C-terminal or internal deletions). Fragments orportions in which only contiguous amino acids have been deleted or inwhich non-contiguous amino acids have been deleted are also envisioned.Generally, mutants or derivatives of the presently disclosed peptidesinclude natural or artificial variants differing by the addition,deletion and/or substitution of one or more contiguous or non-contiguousamino acid residues, or modified peptides in which one or more residuesis modified, and mutants comprising one or more modified residues.

Generally, a functional variant of SEQ ID NO: 1 or SEQ ID NO:2 thereofhas an amino acid sequence which is at least about 80% identical, atleast about 81% identical, at least about 82% identical, at least about83% identical, at least about 84% identical, at least about 85%identical, at least about 86% identical, at least about 87% identical,at least about 88% identical, at least about 89% identical, at leastabout 90% identical, at least about 91% identical, at least about 92%identical, at least about 93% identical, at least about 94% identical,at least about 95% identical, at least about 96% identical, at leastabout 97% identical, at least about 98% identical, or at least about 99%identical to SEQ ID NO:1 or SEQ ID NO:2 over the length of the variant.

Generally, an amino acid sequence that has percent identity to thepresently disclosed sequences has at least about 80% identical, at leastabout 81% identical, at least about 82% identical, at least about 83%identical, at least about 84% identical, at least about 85% identical,at least about 86% identical, at least about 87% identical, at leastabout 88% identical, at least about 89% identical, at least about 90%identical, at least about 91% identical, at least about 92% identical,at least about 93% identical, at least about 94% identical, at leastabout 95% identical, at least about 96% identical, at least about 97%identical, at least about 98% identical, or at least about 99% identicalto the presently disclosed sequences over the length of the variant.

In some embodiments, the presently disclosed subject matter provides anisolated peptide comprising an amino acid sequence at least 85%identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), wherein the peptideexhibits anti-angiogenic, anti-vascular permeability, anti-tumorigenesisand/or anti-lymphangiogenic properties. In other embodiments, thepresently disclosed subject matter provides a composition comprising apharmaceutically acceptable carrier and an effective amount of theisolated peptide comprising an amino acid sequence at least 85%identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), wherein the peptideexhibits anti-angiogenic, anti-vascular permeability, anti-tumorigenesisand/or anti-lymphangiogenic properties. In further embodiments, thepresently disclosed subject matter provides a kit comprising an isolatedpeptide comprising an amino acid sequence at least 85% identical toLRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), wherein the peptide exhibitsanti-angiogenic, anti-vascular permeability, anti-tumorigenesis and/oranti-lymphangiogenic properties. In still further embodiments, thepresently disclosed subject matter provides a nanoparticle ormicroparticle comprising an isolated peptide comprising an amino acidsequence at least 85% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1),wherein the peptide exhibits anti-angiogenic, anti-vascularpermeability, anti-tumorigenesis and/or anti-lymphangiogenic properties.

Further, the presently disclosed peptides can be modified to make themless susceptible to proteolysis. For example, they can be truncated tothe minimal potent sequence. Such truncation is important to limitbinding to other receptors that would dilute the effectiveconcentration, as well as lead to unexpected side effects. Suchtruncation also opens up the possibility to create a single multimodalpeptide out of multiple short peptides, each of which targetsangiogenesis, lymphangiogenesis and tumorigenesis by a differentmechanism. Multimodal treatment is very important to reduce theincidence of drug resistance, because it is less likely that the tumorwill be able to mount a successful resistance when attackedsimultaneously from multiple fronts.

In addition, the presently disclosed peptides with different sequencescan be used together in one composition or method. There may becompositions or methods where multiple types of the presently disclosedpeptides allow better prevention or reduction of angiogenesis, vascularpermeability, tumorigenesis and/or lymphangiogenesis. Therefore, insteadof a composition with a single multimodal peptide, a composition may becomprised of multiple types of isolated peptides that are not covalentlybound together.

Further, in some embodiments, the presently disclosed peptides aretri-fluoro acetate (TFA) salts. For use in humans, however, the TFAsalts can be modified to acetate salts or other pharmaceuticallyacceptable salts.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compositions which are prepared with relatively nontoxic acidsor bases, depending on the particular substituent moieties found on thecompositions described herein. When compositions of the presentdisclosure contain relatively acidic functionalities, base additionsalts can be obtained by contacting the neutral form of suchcompositions with a sufficient amount of the desired base, either neator in a suitable inert solvent. Examples of pharmaceutically acceptablebase addition salts include sodium, potassium, calcium, ammonium,organic amino, or magnesium salt, or a similar salt. When compositionsof the present disclosure contain relatively basic functionalities, acidaddition salts can be obtained by contacting the neutral form of suchcompositions with a sufficient amount of the desired acid, either neator in a suitable inert solvent. Examples of pharmaceutically acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compositionsof the present disclosure contain both basic and acidic functionalitiesthat allow the compositions to be converted into either base or acidaddition salts.

In addition, it is possible to increase the half-lives of the peptidesby conjugating the peptides to certain compositions. For example, it ispossible to conjugate the peptides to catalytic antibodies or topolymers, such as polyethylene glycol (PEG), to increase theirhalf-lives and/or promote self-assembly into particles.

In some embodiments, the presently disclosed subject matter provides acomposition comprising a pharmaceutically acceptable carrier and aneffective amount of a presently disclosed peptide, or a functionalvariant thereof. In other embodiments, the presently disclosed subjectmatte provides a kit comprising a presently disclosed isolated peptide,or a functional variant thereof. The amount of peptide can vary widely,but generally the amount is sufficient to perform at least one of thepresently disclosed methods.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude, but is not limited to, water, saline, dextrose solutions, humanserum albumin, liposomes, hydrogels, microparticles and nanoparticles.The use of such media and agents for pharmaceutically activecompositions is well known in the art, and thus further examples andmethods of incorporating each into compositions at effective levels neednot be discussed here. Such compositions also can include coatings,antibacterial and/or fungal agents, and any other ingredient that isbiologically tolerable.

In general, the “effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, and thelike.

In general, a presently disclosed kit contains some or all of thecomponents, reagents, supplies, and the like to practice a methodaccording to the presently disclosed subject matter. In a kit comprisingan isolated peptide or a functional variant thereof according to thepresently disclosed subject matter, the kit typically comprises aneffective amount of peptide to prevent, delay, reduce, or treat adisease related to angiogenesis, lymphangiogenesis, vascularpermeability, and/or tumorigenesis. In one embodiment, a kit comprisesat least one container (e.g. a vial, tube, or ampoule) comprising anisolated peptide of the presently disclosed subject matter. Typically,the isolated peptide or peptides will be supplied in one or morecontainer, each container containing an effective amount of isolatedpeptide to allow a change in angiogenesis, lymphangiogenesis, vascularpermeability, and/or tumorigenesis to occur.

B. Representative Biodegradable Delivery Platforms

In some embodiments, the presently disclosed peptides are effective whenloaded onto or into, or otherwise associated with, nano- ormicroparticles. Accordingly, in come embodiments, the presentlydisclosed subject matter provides a nanoparticle or microparticlecomprising a presently disclosed peptide, or a functional variantthereof.

In particular embodiments, the nanoparticle or microparticle comprisespoly(lactic-co-glycolic acid) (PLGA) and/or PLGA-polyethylene glycol(PEG). In some embodiments, about 2% to about 5% by mass of the isolatedpeptide is loaded onto or into PLGA and/or PLGA-PEG nanoparticles ormicroparticles. In yet other embodiments, about 6% to about 10% by massof the isolated peptide is loaded onto or into the PLGA and/or PLGA-PEGnanoparticles or microparticles.

Further, in other embodiments, certain polymer formulations,microparticles, nanoparticles, and the like, which are suitable for usewith the presently disclosed subject matter are disclosed inInternational PCT Patent Application Publication No. WO/2012/0128782 for“Multicomponent Degradable Cationic Polymers.” International PCT PatentApplication Publication No. WO/2012/0114759 for “Peptide/ParticleDelivery Systems,” International PCT Patent Application Publication No.WO/2014/066811 for “Bioreducible Poly(beta-amino ester)s for siRNADelivery,” and International PCT Patent Application Publication No.WO/2014/066898 for “A Layer-by-Layer Approach to Co-deliver DNA andsiRNA via AuNPs: a Potential Platform for Modifying Release Kinetics,”all to Green et al., and each of which is incorporated herein byreference in its entirety.

As used herein, the term “poly(beta-amino ester) (PBAE)” can refer to acompound of the general formula:

wherein:

n is an integer from 1 to 10,000;

R comprises a backbone of a diacrylate selected from the groupconsisting of:

R′ comprises a side chain derived from a compound selected from thegroup consisting of:

R″ comprises an end group derived from a compound selected from thegroup consisting of

In such embodiments, the polymer compositions can be designated as, forexample, B6-S5-E7 or 657, in which R is B6, R′ is S5, and R″ is E7.

Accordingly, in some embodiments, the nanoparticle or microparticlecomprises a poly(beta-amino ester) (PBAE) and/or PBAE-PEG. In particularembodiments, the nanoparticle or microparticle comprises about 1% toabout 5% by mass of the isolated peptide loaded onto or into the PBAEand/or PBAE-PEG nanoparticles or microparticles. In yet otherembodiments, the nanoparticle or microparticle comprises about 6% toabout 10% by mass of the isolated peptide loaded onto or into the PBAEand/or PBAE-PEG nanoparticles or microparticles.

In yet other embodiments, the nanoparticle or microparticle comprises acombination of a poly(beta-amino ester) (PBAE), PLGA, and PEG. Inparticular embodiments, the nanoparticle or microparticle comprisesabout 1% to about 5% by mass of the isolated peptide loaded onto or intoparticles comprising a combination of a poly(beta-amino ester) (PBAE),PLGA, and PEG. In still other embodiments, the nanoparticle ormicroparticle comprises about 6% to about 10% by mass of the isolatedpeptide loaded onto or into particles comprising a combination of apoly(beta-amino ester) (PBAE), PLGA, and PEG.

As used herein, the term “nanoparticle,” refers to a particle having atleast one dimension in the range of about 1 nm to about 1000 nm,including any integer value between 1 nm and 1000 nm (including about 1,2, 5, 10, 20, 50, 60, 70, 80, 90, 100, 200, 500, and 1000 nm and allintegers and fractional integers in between). In some embodiments, thenanoparticle has at least one dimension, e.g., a diameter, of about 100nm. In some embodiments, the nanoparticle has a diameter of about 200nm. In other embodiments, the nanoparticle has a diameter of about 500nm. In yet other embodiments, the nanoparticle has a diameter of about1000 nm (1 μm). In such embodiments, the particle also can be referredto as a “microparticle”. Thus, the term “microparticle” includesparticles having at least one dimension in the range of about onemicrometer (μm), i.e., 1×10⁻⁶ meters, to about 1000 μm. The term“particle” as used herein is meant to include nanoparticles andmicroparticles. In some embodiments, the microparticle is between 1-5μm. In other embodiments, the microparticle is 3-10 μm. In some otherembodiments, the microparticle is 10-20 μm. In further embodiments, themicroparticles and nanoparticles are spherical in shape. In stillfurther embodiments, the microparticles and nanoparticles have anon-spherical shape. In some embodiments, the particles have anellipsoidal shape with an aspect ratio of the long axis to the shortaxis between 2 and 10.

In some embodiments, the three-dimensional microparticle or nanoparticlecomprises a material having one or more of the followingcharacteristics: (i) one or more degradable linkages; (ii) a stretchablemodulus; and (iii) a glass transition temperature such that the materialcomprising the three-dimensional microparticle or nanoparticle is asolid at room temperature and/or body temperature. In other embodiments,the degradable linkage is selected from the group consisting of an esterlinkage, a disulfide linkage, an amide linkage, an anhydride linkage,and a linkage susceptible to enzymatic degradation.

In particular embodiments, the microparticle or nanoparticle comprises abiodegradable polymer or blends of polymers selected from the groupconsisting of poly(lactic-co-glycolic acid) (PLGA), poly(beta-aminoester) (PBAE), polycaprolactone (PCL), polyglycolic acid (PGA),polylactic acid (PLA), poi acrylic acid) (PAA), poly-3-hydroxybutyrate(P3HB) and poly(hydroxybutyrate-co-hydroxyvalerate). In otherembodiments, nondegradable polymers that are used in the art, such aspolystyrene, are blended with a degradable polymer or polymers fromabove to create a copolymer system. Accordingly, in some embodiments, anondegradable polymer is blended with the biodegradable polymer.

As used herein, “biodegradable” compositions are those that, whenintroduced into cells, are broken down by the cellular machinery or byhydrolysis into components that the cells can either reuse or dispose ofwithout significant toxic effect on the cells (i.e., fewer than about20% of the cells are killed when the components are added to cells invitro). The components preferably do not induce inflammation or otheradverse effects in vivo. In certain preferred embodiments, the chemicalreactions relied upon to break down the biodegradable compositions areuncatalyzed.

In some other embodiments, the microparticle or nanoparticle isbiocompatible. The term “biocompatible”, as used herein is intended todescribe compositions that are not toxic to cells. Compositions are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and their administration in vivo does notinduce inflammation or other such adverse effects.

It will be appreciated by one of ordinary skill in the art thatnanoparticles and microparticles suitable for use with the presentlydisclosed methods can exist in a variety of shapes, including, but notlimited to, spheroids, rods, disks, pyramids, cubes, cylinders,nanohelixes, nanosprings, nanorings, rod-shaped particles, arrow-shapedparticles, teardrop-shaped particles, tetrapod-shaped particles,prism-shaped particles, and a plurality of other geometric andnon-geometric shapes.

C. General Terms

For clarity, other general terms are described below. Sequence identityis typically measured using sequence analysis software (for example,Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs).Such software matches identical or similar sequences by assigningdegrees of homology to various substitutions, deletions, and/or othermodifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. In an exemplary approach to determining the degree ofidentity, a BLAST program may be used, with a probability score betweene⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window, and can take into considerationadditions, deletions and substitutions. When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (for example,charge or hydrophobicity) and therefore do not deleteriously change thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have sequence similarity. Approaches for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, for example,according to the algorithm of Meyers and Miller, Computer Applic. Biol.Sci., 4: 11-17, 1988, for example, as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

“Percentage of sequence identity” means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions, substitutions, or deletions (i.e., gaps)as compared to the reference sequence (which does not compriseadditions, substitutions, or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The term “substantial identity” or “homologous” in their variousgrammatical forms in the context of polynucleotides means that apolynucleotide comprises a sequence that has a desired identity, forexample, at least 60% identity, preferably at least 70% sequenceidentity, more preferably at least 80%, still more preferably at least90% and even more preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 85%, 90%, and even more preferably at least 95%.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 5, 10, or 15 amino acids,preferably at least about 20 amino acids, more preferably at least about25 amino acids, and even more preferably about 35 amino acids, about 50amino acids, about 100 amino acids, or about 150 amino acids.

II. Methods of Treating Angiogenesis- and Lymphangiogenesis-DependentDiseases A. Representative Embodiments

In some embodiments, the presently disclosed peptides exhibitanti-angiogenic anti-lymphangiogenic, anti-tumorigenic, and/oranti-vascular permeability properties. Angiogenesis refers to the growthof new blood vessels originating from existing blood vessels.Lymphangiogenesis refers to the formation of lymphatic vessels de novoor from pre-existing lymphatic vessels, in a method believed to besimilar to blood vessel development or angiogenesis. Tumorigenesisrefers to the formation of a tumor. Vascular permeability refers to theproperty of blood microvascular walls including blood capillary wallsthat allows for the selective exchange of substances.

In some embodiments, the presently disclosed subject matter provides amethod for inhibiting angiogenesis, lymphangiogenesis, vascularpermeability and/or tumorigenesis involving a cell. The method comprisescontacting a cell with a presently disclosed isolated peptide or afunctional variant thereof in an amount sufficient to inhibitangiogenesis, lymphangiogenesis, vascular permeability and/ortumorigenesis of the cell. The contacting of the cell may result in aninhibition of adhesion, migration, proliferation, and/or tube formationinvolving the cell. In a particular embodiment, the cell is anendothelial cell.

In other embodiments, the method for inhibiting angiogenesis,lymphangiogenesis, vascular permeability and/or tumorigenesis involvinga cell comprises: contacting the cell with an isolated peptidecomprising an amino acid sequence at least 85% identical toLRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), in an amount sufficient to inhibitangiogenesis, lymphangiogenesis, vascular permeability and/ortumorigenesis involving the cell. In further embodiments, contacting thecell results in an inhibition of adhesion, migration, proliferation,and/or tube formation involving the cell. In a particular embodiment,the cell is selected from the group consisting of an endothelial cell, amicrovascular cell, and a lymphatic cell. In other embodiments, the cellis found in adipose tissue and the method reduces or prevents obesity.In still other embodiments, the cell is a transplanted cell and themethod reduces or prevents tissue and/or organ rejection aftertransplantation of the cell. By “transplantation of the cell”, it ismeant that the cell is moved from one body to another body.

In some embodiments, the isolated peptide is loaded onto or into ananoparticle or microparticle before contacting the cell. In otherembodiments, the nanoparticle or microparticle comprises PLGA and/orPLGA-PEG.

The methods of the presently disclosed subject matter can be practicedin vivo as either a therapeutic method for treating a disease ordisorder involving angiogenesis, lymphangiogenesis, vascularpermeability and/or tumorigenesis or as a prophylactic method to preventangiogenesis, lymphangiogenesis, vascular permeability and/ortumorigenesis. Likewise, the method can be practiced in vitro as aresearch tool to study the effects of angiogenesis, lymphangiogenesis,vascular permeability and/or tumorigenesis on a cell. The method alsocan be practiced ex vivo for therapeutic or research purposes.

“Contacting” means any action that results in at least one isolatedpeptide of the presently disclosed subject matter physically contactingat least one cell. It thus may comprise exposing the cell(s) to theisolated peptide in an amount sufficient to result in contact of atleast one isolated peptide with at least one cell. The method can bepracticed in vitro or ex vivo by introducing, and preferably mixing, theisolated peptide and cells in a controlled environment, such as aculture dish or tube. The method can be practiced in vivo, in which casecontacting means exposing at least one cell in a subject to at least oneisolated peptide of the presently disclosed subject matter, such asadministering the isolated peptide to a subject via any suitable route.According to the presently disclosed subject matter, contacting maycomprise introducing, exposing, and the like, the isolated peptide at asite distant to the cells to be contacted, and allowing the bodilyfunctions of the subject, or natural (e.g., diffusion) or man-induced(e.g., swirling) movements of fluids to result in contact of theisolated peptide and cell(s).

In some embodiments, the presently disclosed subject matter provides amethod for treating a subject suffering from a disease related toangiogenesis, lymphangiogenesis, vascular permeability and/ortumorigenesis, or to prevent or delay a subject from developing adisease related to angiogenesis, lymphangiogenesis, vascularpermeability and/or tumorigenesis, the method comprising: administeringto the subject a presently disclosed isolated peptide, or a functionalvariant thereof, in an amount sufficient to treat, delay, or prevent thedisease in the subject.

In particular embodiments, the presently disclosed subject matterprovides a method for treating a subject suffering from a diseaserelated to angiogenesis, lymphangiogenesis, vascular permeability,and/or tumorigenesis or to prevent or delay a subject from developing adisease related to angiogenesis, lymphangiogenesis, vascularpermeability, and/or tumorigenesis, the method comprising: administeringto the subject an isolated peptide comprising an amino acid sequence atleast 85% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), in an amountsufficient to treat, delay, or prevent the disease in the subject.

In some embodiments, the subject is human. In other embodiments, thesubject is nonhuman.

Representative diseases include those diseases that are angiogenesis-,lymphangiogenesis-, tumorigenesis-, and or vascularpermeability-dependent. Accordingly, in some embodiments, the disease isa cancer. In other embodiments, the cancer is selected from the groupconsisting of breast, lung, glioblastoma, renal cell, hepatic cell,head, and neck cancer. In still other embodiments, the method inhibitsangiogenesis, lymphangiogenesis, vascular permeability, and/ortumorigenesis in or surrounding a tumor. In further embodiments, thetumor is a primary tumor or an established metastasized tumor. In stillfurther embodiments, the method inhibits the establishment of metastatisor inhibits further metastasis of the cancer. In other embodiments, themethod inhibits dissemination of tumor cells through the blood and/orlymphatic vasculature.

In some embodiments, the method inhibits lymphangiogenesis, angiogenesisand/or tumorigenesis perioperatively to reduce or prevent tumor regrowthand metastasis. In other embodiments, the method inhibitslymphangiogenesis, angiogenesis and/or tumorigenesis postoperatively,after surgical complete or partial excision of the primary tumor,thereby reducing or preventing tumor regrowth and metastasis. Therefore,in some embodiments, the method inhibits lymphangiogenesis, angiogenesisand/or tumorigenesis perioperatively and/or postoperatively.

In still further embodiments, the method inhibits lymphangiogenesis,thereby reducing or preventing tissue and organ rejection aftertransplantation, e.g., with skin grafts, bone grafts, and other tissuesand organs.

In some embodiments, the method inhibits angiogenesis and/orlymphangiogenesis in adipose tissue thereby reducing or preventingobesity.

In some embodiments, the disease is related to ocular angiogenesis ordiabetic retinopathy. In other embodiments, the disease is selected fromthe group consisting of age-related macular degeneration, macular edema,neovascular glaucoma, proliferative diabetic retinopathy, andretinopathy of prematurity.

In further embodiments, the isolated peptide is loaded into or onto ananoparticle or microparticle before administering to the subject. Instill further embodiments, the nanoparticle or microparticle comprisesPLGA and/or PLGA-PEG. In other embodiments, about 2% to about 5% by massof the isolated peptide is loaded onto or into PLGA nanoparticles ormicroparticles. In yet other embodiments, about 6% to about 10% by massof the isolated peptide loaded onto or into the PLGA and/or PLGA-PEGnanoparticles or microparticles.

In some embodiments, the isolated peptide is administered in combinationwith at least one other anti-angiogenesis agent. In particularembodiments, the at least one other anti-angiogenesis agent is selectedfrom the group consisted of aflibercept, ranibizumab, bevacizumab, andcombinations thereof. In certain embodiments, the presently disclosedsubject matter provides for the use of the isolated peptides in thetreatment of a disease associated with angiogenesis, lymphangiogenesis,tumorigenesis, and/or vascular permeability. The use is in particularfor in vivo therapeutic or prophylactic methods of inhibitingangiogenesis, lymphangiogenesis, tumorigenesis, and/or vascularpermeability. Certain embodiments provide for the use of the isolatedpeptides in the preparation of compositions for medical use, such aspharmaceutical or therapeutic compositions. In general, use of theisolated peptides is in combining them with other substances to makemedicinal compositions.

The peptides according to the disclosure are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, andfrom 5 to 40 mg per day are examples of dosages that may be used. Anon-limiting dosage is 10 to 30 mg per day. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the subject to be treated, the body weight of the subjectto be treated, and the preference and experience of the attendingphysician.

B. General Terms

By “disease” is meant any condition, dysfunction or disorder thatdamages or interferes with the normal function of a cell, tissue, ororgan.

A “cancer” in an animal refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, for example,uncontrolled proliferation, loss of specialized functions, immortality,significant metastatic potential, significant increase in anti-apoptoticactivity, rapid growth and proliferation rate, and certaincharacteristic morphology and cellular markers. In some circumstances,cancer cells will be in the form of a tumor; such cells may existlocally within an animal, or circulate in the blood stream asindependent cells, for example, leukemic cells.

By “blood vessel formation” is meant the dynamic process that includesone or more steps of blood vessel development and/or maturation, such asangiogenesis, vasculogenesis, formation of an immature blood vesselnetwork, blood vessel remodeling, blood vessel stabilization, bloodvessel maturation, blood vessel differentiation, or establishment of afunctional blood vessel network.

By “vasculogenesis” is meant the development of new blood vesselsoriginating from stem cells, angioblasts, or other precursor cells.

By “blood vessel stability” is meant the maintenance of a blood vesselnetwork.

By “neoplasia” is meant a disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. Solid tumors, hematological disorders, andcancers are examples of neoplasias.

A “tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reduce” is meant a decrease in a parameter (e.g., blood vesselformation) as detected by standard art known methods, such as thosedescribed herein. As used herein, reduce includes a 10% change,preferably a 25% change, more preferably a 40% change, and even morepreferably a 50% or greater change.

The subject treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes, such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein.

In therapeutic and/or diagnostic applications, the compositions of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000). To aid in bioavailability, the compositions of the disclosuremay be delivered in a nano- or micro-particles.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example, but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Pharmaceutically acceptable saltsinclude, for example, acetate, benzoate, bromide, carbonate, citrate,gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate,pamoate (embonate), phosphate, salicylate, succinate, sulfate, ortartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articullar, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions, such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompositions herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular,those formulated as solutions, may be administered parenterally, such asby intravenous injection. The compositions can be formulated readilyusing pharmaceutically acceptable carriers well known in the art intodosages suitable for oral administration. Such carriers enable thecompositions of the disclosure to be formulated as tablets, pills,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompositions into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compositions with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (scC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compositions may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols (PEGs). In addition, stabilizers may be added.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, ±100% insome embodiments ±50%, in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The following Examples are offered by way ofillustration and not by way of limitation.

Example 1 Materials and Methods

Growth of Cells in Cell Culture.

For the growth of cells in cell culture, human umbilical veinendothelial cells (HUVEC), microvascular endothelial cells (MEC), andlymphatic endothelial cells (LEC) were purchased from Lonza andmaintained according to the manufacturer's recommendation usingEndothelial Basal Media (EBM-2) supplemented with the Bullet Kit (EGM-2,Lonza). The MEC and LEC were propagated in Microvascular EndothelialCell Growth Medium-2 (EGM-2MV, Lonza). Breast cancer cells, MDA-MB-231were supplied by Dr. Zaver Bhujwalla (JHMI, Radiology and Oncology). Thecells were propagated in RPMI-1640 medium (Gibco, Carlsbad, Calif.)supplemented with 10% FBS and antibiotics (1% penicillin/streptomycin).Cells were maintained under standard conditions of 37° C. and 5% CO₂ andthe passage numbers of all used cells were between 2 and 7.

Peptide Synthesis.

Peptides were synthesized using solid state synthesis and were suppliedas TFA salts with an amidated C-terminus and an amine at the N-terminus(New England Peptide, Gardner, Mass.). The purity of the peptideswas >95% and the suppliers provided product characterization (MALDI-TOF,HPLC/MS, and HPLC traces) as proof of MW and purity accuracy. Peptideswere solubilized in 5% DMSO and water due to their hydrophobic profile.The pH of solubilized peptides was checked and found to be around pH 7.For all experiments the DMSO % was maintained at non-toxic threshold(determined by toxicity curves of DMSO on cells) with a final DMSOpercentage (<0.2%), which was used as control in all experiments.

Proliferation Assay.

A colorimetric based proliferation assay using WST-1 (Roche,11644807001) proliferation reagent was carried out using HREC cells,HUVEC cells, and the breast cancer cells MDA-MB-231, MCF-7, and SUM149.2000 cells/well were plated in 96-well plates and allowed to adhereovernight. On the following day, the media was exchanged with fullysupplemented media containing peptides or equivalent DMSO vehicle forthe controls. Three days later, the media containing the peptides wasreplaced with serum-free EBM-2 media containing WST-1 reagent and theplates were incubated for four hours as per the manufacturer'srecommendations. Changes in color, due to the formazan dye resulted fromthe cleavage of the tetrazolium salt WST-1 by the mitochondrialsuccinate-tetrazolium reductase, were read on a Victor V fluorescenceplate reader (Perkin Elmer, Mass.) by measuring the absorbance at 450nm. Dose response curves of percent live cells (in comparison tountreated cells, but incubated in complete media with 0.2% DMSO) werecreated. Assays were performed in at least two independent replicatesand each replicate was performed using three experimental triplicates.

Migration Assay.

The inhibitory potential of the peptides was measured using a real timemigration assay system based on electrical impedance (RT-CIM, ACEABiosciences, CA). CIM 16 well plates (Roche, 05665817001) are composedof a top and bottom chamber separated by a microporous (8 μm)polycarbonate membrane. The membrane was coated with fibronectin (20μg/mL), and 45,000 cells/well in serum free media with or withoutpeptides were added to the top compartment. Media with chemoattractant(i.e., fully supplemented EBM-2) was added to the bottom compartment ofthe chamber and the plate was incubated at 37° C. for 20 hours. Thesensors integrated on the bottom side of the membrane monitor andcontinuously record changes in impedance as the cells move through themembrane. The RT-CIM technology allows for easy quantification of cellmigration by monitoring the cell index derived from the measuredimpedances. Assays were performed in at least two independent replicatesand each replicate was performed using two experimental duplicates.Breast cancer cells MDA-MB-231 are not suitable for the RT-CIM typeexperiments due to their thin elongated phenotype, thus the inhibitionof migration was investigated using a wound healing type assay. Thisassay was performed using the Oris Pro Migration assay (PlatypusTechnologies, CMA 1.101). Briefly, 25,000 cells/well in full media wereadded to the 96 well plate containing stoppers to block migration ofcells to the center region of the wells. Cells were allowed to adherefor 4 hours, after which the stoppers were removed. Cells were washedone time with PBS and fully supplemented media, with or withoutcompound, was added to the wells. After 18 hours, cells were stainedwith calcein AM (0.5 μg/mL) (Invitrogen, CA) and the cells that migratedto the center of the well were imaged using a Nikon microscope (EclipseT-100); images were acquired with the CCD Sensicam mounted on a Nikonmicroscope (Cooke Company, Mich.). The detection of the cells thatmigrated into the previously restricted region was possible due to theaddition of a detection mask at the bottom of the plate, whichobstructed from measurement cells that did not migrate.

Wound Healing Assay.

Breast cancer cells MDA-MB-231 are not suitable for the RT-CIM typeexperiments due to their thin elongated phenotype, thus the inhibitionof migration was investigated using a wound healing type assay. Thisassay was performed using the Oris Pro Migration assay (PlatypusTechnologies, CMA 1.101). Briefly, 25,000 cells/well in full media wereadded to the 96 well plate containing stoppers to block migration ofcells to the center region of the wells. Cells were allowed to adherefor 4 hours, after which the stoppers were removed. Cells were washedone time with PBS and fully supplemented media, with or withoutcompound, was added to the wells. After 18 hours, cells were stainedwith calcein AM (0.5 μg/mL) (Invitrogen, CA) and the cells that migratedto the center of the well were imaged using a Nikon microscope (EclipseT-100); images were acquired with a CCD Sensicam mounted on a Nikonmicroscope (PCO-TECH, Inc., Romulus, Mich.). The detection of the cellsthat migrated into the previously restricted region was possible due tothe addition of a detection mask at the bottom of the plate, whichobstructed from measurement cells that did not migrate.

Adhesion Assays.

Similar to the migration assays, the inhibition activity of the peptidein cellular adhesion was assessed using the RT-CIM technology. In thiscase, 25,000 cells/well were plated in 16 wells E-plates (Roche, BaselSwitzerland) in the presence or absence of the peptide. The adhesion wasmonitored over time (3 hours) by measuring changes in the electricalimpedance, which is a direct measure of the cells adhering on theelectrodes. Assays were performed in at least two independent replicatesand each replicate was performed using two experimental duplicates.

Tube Formation Inhibition Assays.

The compositions also were tested for their ability to inhibit tubeformation, a process critical in angiogenesis. Endothelial cellsspontaneously form a network of tubes when plated on extracts ofextracellular matrix. This in vitro assay combines aspects of adhesionand migration and it is routinely used in angiogenesis research(Oliveira-Ferrer et al., 2008). The ability to inhibit tube formation isa comprehensive assessment of the anti-angiogenic potential. Theprotocol was described by Arnaoutova et al. (2009) and it consists ofplating HUVEC on top of basement membrane extract. After incubation at37° C., the cells naturally rearrange themselves in a network of tubes.Thus, 50 μL/well of Matrigel (BD Biosciences, San Jose, Calif.) wasplated in a cold 96 well plate and incubated at 37° C. for 30 min forpolymerization. 15,000 cells/well were added to the top of the gel andincubated in complete media in the presence or absence of peptide for 19hours. Images were captured using the CCD Sensicam mounted on a Nikonmicroscope (Eclipse T-100). Assays were performed in at least twoindependent replicates and each replicate was performed using threeexperimental replicates and one image of a randomly chosen field wasacquired per well.

Tumor Xenografts.

Orthotopic breast tumors were initiated in SCID mice using humantriple-negative breast cancer cells MDA-MB-231. 2×10⁶ cells per 100 μLaliquot of single cell suspension were injected in the breast mammaryfat pad. Tumors reached volumes of 75-100 mm³ in approximately 14-21days. Mice were randomized and arranged in groups (8 mice per group)with similar tumor volumes (no statistical difference among averages)and treatment was commenced. The peptide was administered once per dayintraperitoneally (i.p.) at a dose of 10 mg/kg. Tumors were measuredevery fourth day using calipers and the tumor volume was calculatedusing the formula V=ab²/2, where “a” is the larger and “b” is thesmaller diameter.

TCM-Induced Metastasis Model.

Before tumor inoculation, athymic nude mice (female 5-6 weeks, 18-20 g)were treated with 50 μL TCM or SFM subcutaneously through the scruff for2 weeks. After 2 weeks of TCM treatment, MDA-MB-231-luc-D3H2LN tumorxenografts were established in the same animals; the cells (2×10⁶) weremixed with 50 μL complete media (RPMI-1640 supplemented with 10% FBS),and 50 μL Matrigel (High Concentrated, BD Biosciences) and injected intothe upper inguinal mammary fat pad of the animals under anesthesia (50mg/kg ketamine+5 mg/kg acepromazine in PBS, 50 μL/animal,intraperitoneally dosed). The primary tumor size was measured by using acaliper, and the volume w calculated, using the formula: V=0.52×a×b²,where ‘a’ is the long axis, and ‘b’ is the short axis of the tumor.Animals were also imaged every week to track anterior tumor metastases,using the IVIS Xenogen 200 optical imager (Xenogen, Alameda, Calif.)after intraperitoneal injection of d-luciferin (Caliper 150 mg/kg). 100μL D-luciferin (twice diluted) was intraperitoneally dosed on both sidesof the abdomen (total 200 μL/animals) to prevent i.p. injection failure.After 4 weeks, the axillary and brachial LN (lymph nodes), the lungs,and the brain were harvested and bathed in D-luciferin solution for 3min and placed in the IVIS imager to detect metastases ex vivo.Luciferase-mediated photon flux was quantified by using Living Image® 3DAnalysis (Xenogen), and the average photon flux was obtained from eightlungs, eight brains, and 14-16 LNs as described before. In case ofanimals showing intra-abdominal metastases, ex vivo images of abdominalorgans, including stomach, spleen, kidney, liver, and intestine, wereobtained using the IVIS imager.

Mouse Model of Choroidal Neovascularization (CNV).

Laser photocoagulation-induced rupture of Bruch's membrane was used togenerate CNV. Briefly, 4-5 week old female C57BL/6J mice wereanesthetized with xylazine hydrochloride (10 mg/kg) and ketaminehydrochloride (50 mg/kg) and the pupils were dilated with 1% tropicamide(Alcon Labs, Inc., Fort Worth, Tex., USA). Three burns of 532-nm diodelaser photocoagulation (75 μm spot size, 0.1 s duration, 120 mW) weredelivered to each retina using the slit lamp delivery system of anOcuLight GL Photocoagulator (Index, Mountain View, Calif., USA) and ahand-held cover slide as a contact lens. Burns were performed in the 9,12 and 3 o'clock positions of the posterior pole of the retina.Production of a bubble at the time of laser photocoagulation, whichindicates rupture of Bruch's membrane, is an important factor inobtaining CNV, so only burns in which a bubble was produced wereincluded in the study. Treatments were begun seven days after laserphotocoagulation and included intravitreous injections of 0.1% of SP2043(SEQ ID NO: 1) (1 μg in a volume of 1 μL), 1% (10 m in a volume of 1 μLof SP2043 (SEQ ID NO: 1)) or vehicle under a dissecting microscope witha Harvard Pump Microinjection System (Harvard Apparatus, Holliston,Mass.) and pulled glass micropipettes. Some mice were euthanized forbaseline measurement. Fourteen days after laser, the remaining mice wereused to measure the amount of CNV at Bruch's membrane rupture sites. Twoweeks after rupture of Bruch's membrane, mice were anesthetized andperfused with fluorescein-labeled dextran (2×10⁶ average mol wt,Sigma-Aldrich, St. Louis, Mo., USA) and choroidal flat mounts wereprepared as described previously. Briefly, the eyes were removed, fixedfor 1 h in 10% phosphate-buffered formalin, and the cornea and lens wereremoved. The entire retina was carefully dissected from the eyecup, andthen radial cuts were made from the edge of the eyecup to the equator inall four quadrants and flat-mounted in Aquamount (Polysciences,Warrington, Pa.). Flat mounts were examined by fluorescence microscopyusing an Axioskop microscope (Zeiss, Thornwood, N.Y., USA) and imageswere digitized using a 3 CCD color video camera (IK-TU40A, Toshiba,Tokyo, Japan) and a frame grabber. Image-Pro Plus software (MediaCybernetics, Silver Spring, Md., USA) was used to measure the area ofeach CNV lesion. Statistical comparisons were made using ANOVA andBonferroni.

CNV Regression Model.

Bruch's membranes of both eyes of C57BL/6J mice were ruptured with alaser after anesthetizing the mice and dilating their pupils with 1%tropicamide. Seven days later, some mice were sacrificed to establishbaseline neovascularization. At that time, 1 μg of SP2043 (SEQ ID NO: 1)was administered in one eye and vehicle in the fellow eye of other mice.Seven days later, all animals were perfused with fluorescein-labeleddextran, eyes removed, the retina dissected and flatmounted after makingradial cuts, and fluorescence was measured. The images were digitizedusing a 3-color charge-coupled device video camera and a frame grabberand the area of hypervascularization was quantified by image-analysissoftware.

Transgenic Mice with VEGF-Induced Neovascularization.

At postnatal day 14, hemizygous rho/VEGF mice were given an intraocularinjection of 1 μL of 5% DMSO/water, 1 μL of 5% DMSO/water containing 0.1μg or 1 μg of SP2043 (SEQ ID No: 1) in one eye. Intraocular injectionswere done under a dissecting microscope with a Harvard PumpMicroinjection System (Harvard Apparatus, Holliston, Mass.) and pulledglass micropipettes. At postnatal day 21, the total area of subretinalneovascularization (NV) per eye was quantified. Briefly, mice wereanesthetized and perfused with 1 mL of PBS containing 25 mg/mL offluorescein-labeled dextran (average molecular weight of 2×10⁶,Sigma-Aldrich, St. Louis, Mo.). The eyes were removed and fixed for 1hour in 10% phosphate-buffered formalin. The cornea and lens wereremoved, and the entire retina was carefully dissected from the eyecup,radially cut from the edge of the retina to the equator in all 4quadrants, and flat-mounted in mounting medium (Aquamount; Polysciences,Warrington, Pa.) with photoreceptors facing upward. The retinas wereexamined by fluorescence microscopy at 200× magnification, whichprovides a narrow depth of field so that when focusing on NV on theouter surface of the retina, the remainder of the retinal vessels areout of focus, allowing easy delineation of the NV. The outer edge of theretina, which corresponds to the subretinal space in vivo, is easilyidentified and therefore there is standardization of focal plane fromslide to slide. Images were digitized using a 3 charged-coupled device(CCD) color video camera and a frame grabber. By using Image analysissoftware (Image-Pro Plus; Media Cybernetics, Silver Spring, Md.), aninvestigator masked with respect to treatment group allowed the softwareto recognize and calculated the total area of subretinal NV per eye aspreviously described.

Vascular Permeability in Mouse Eyes:

Mice were injected intravitreally with 1 μg of SP2043 (SEQ ID NO: 1) inone eye, and vehicle in the fellow eye and vehicle control was injectedin the other eye of adult double transgenic Tet/Opsin/VEGF mice. Threedays later, the mice were given 2 mg/mL doxycycline in the drinkingwater for 3 days. At that time the animals were anesthetized and theirpupils dilated as for standard fundoscopy. The eyes were examined tocompare the number of animals that had retinal detachments under the twotreatments by fundus imaging using Micron II retinal imaging and opticalcoherence tomography imaging using published protocols.

Vascular Permeability Measurement in Rabbit Eyes:

At day 0, the SP2043 peptide (SEQ ID NO:1; 50 μg) was injected into thevitreous of Dutch-belted pigmented rabbit and vehicle was injected intothe fellow eye. On day 3, 50 μg of VEGF (human) was injected into botheyes. On day 10, fluorescein was injected systemically into the rabbitsand 2 h later the fluorescence 5 mm to 8 mm in front of the retina wasmeasured by a Fluoroton Master FM-2 ocular flouorophotomoter.

Materials for Peptide Formulations:

PLGA [poly(D,L-lactide-co-glycolide); threeformulations—lactide:glycolide (65:35), MW 40,000-75,000;lactide:glycolide (72:25), MW 76,000-115,000; lactide:glycolide (85:15),MW 190,000-240,000], DCM [dichloromethane], DMSO [dimethylsulfoxide],and DMF [N,N-dimethylformamide] were purchased from Sigma (St. Louis,Mo.). PVA [poly(vinyl alcohol); Mw 25,000] was purchased fromPolysciences (Warrington, Pa.). PLGA-PEG [methoxy poly(ethyleneglycol)-b-poly(lactide-co-glycolide); PEG:PLGA 5:20 kDa; 1:1 LA:GA) waspurchased from PolySciTech (West Lafayette, Ind.). PBAEs[Poly(beta-amino ester)s] were synthesized as previously described. NaAcbuffer (pH=5) [sodium acetate buffer] was purchased from Invitrogen(Grand Island, N.Y.).

PLGA Microparticles Loaded with Peptide:

PLGA was first dissolved into DCM, at desired concentration (usually 20mg/mL or 40 mg/mL), in a test tube and vortexed to fully dissolve.Peptide stock in DMSO (usually 20 mg/mL) was micropipetted to thePLGA/DCM solution. The mass ratio of peptide to PLGA can vary; a commonformulation is 1:50 peptide:PLGA. For blank microparticle, pipetteequivalent volume of DMSO only was used. The mixture was sonicated withthe test tube on ice. Sonication was performed with an amplitude settingof ‘30’, which equaled approximately 5-10 W, for 20 seconds. Thisprimary emulsion was immediately poured into 50 mL of 1% PVA solutionand homogenized at 3.6-3.8 krpm for 1 minute. The full volume was thentransferred to 100 mL of 0.5% PVA solution and stirred in a chemicalhood for 3 hours. Three wash steps were performed. For each wash step,the microparticle solution was centrifuged at 4° C., 4 krpm, for 5minutes, and then the supernatant was removed. Subsequently, 40 mL ofrefrigerated water was added, the microparticle pellet was resuspendedand the washing steps were repeated. After the last centrifugation step,5 mL of water was added to resuspend the sample. Samples were snapfrozen in liquid nitrogen and immediately placed in a lyophilizer.Following lyophilization, all microparticles were stored at −20° C.

PLGA Nanoparticles Loaded with Peptide:

PLGA was first dissolved into DCM, at desired concentration (usually 20mg/mL or 40 mg/mL), in a test tube and vortexed to fully dissolve.Peptide stock in DMSO (usually 20 m/mL) was micropipetted to thePLGA/DCM solution. The mass ratio of peptide to PLGA can vary; a commonformulation is 1:50 peptide:PLGA. For blank nanoparticle, pipettedequivalent volume of DMSO only was used. The mixture was sonicated withthe test tube on ice. Sonication was performed with an amplitude settingof ‘30’, which equals approximately 5-10 W, for 20 seconds. This primaryemulsion was immediately poured into 50 mL of 1% PVA solution andsonicated at an amplitude setting of ‘60’ for 2 minutes. The full volumewas then transferred to 100 mL of 0.5% PVA solution and stirred in achemical hood for 3 hours. Three wash steps were performed. For eachwash step, the microparticle solution was centrifuged at 4° C., 17 krpm,for 10 minutes, and then the supernatant was removed. Subsequently, 30mL of refrigerated water was added, the microparticle pellet wasresuspended and the washing steps were repeated. After the lastcentrifugation step, 5 mL of water was added to resuspend the sample.Samples were snap frozen in liquid nitrogen and immediately placed in alyophilizer. Following lyophilization, all microparticles were stored at−20° C.

PLGA-PEG Nanoparticles Loaded with Peptide:

PLGA-PEG, PLGA-PEG-maleimide, or PLGA-PEG-conjugated to a ligand wasfirst dissolved in DMF at 10 mg/mL (or other desired concentration).Then peptide (2043, 2043-IRD800; stocks at 20 mg/mL in DMF) was added toPLGA-PEG for final peptide w/w % of 2% (or other desired ratio). Forblank nanoparticles, an equivalent volume of DMF only was added toPLGA-PEG. The PLGA-PEG/peptide/DMF solution was added dropwise toMilli-Q water spinning on a stir plate for a final volume ratio oforganic:aqueous at 1:10. The mixture was spun under a chemical hood for4 hours. Particles were washed twice using Amicon Ultra-15 centrifugetubes (Millipore) at 4° C., for 10 min each spin. Concentrated particleswere stored either in water at 4° C., or lyophilized with differentamounts of sucrose (Sigma-Aldrich).

Polymer Peptide Complexes:

Each type of polymer (PBAEs) and peptide (SP2043; SEQ ID NO:1) wasdiluted in NaAc buffer at varying concentrations depending on desiredPBAE:2043 mass ratios (from 1:1 to 100:1). The PBAE solution waspipetted to the 2043 solution and incubated at room temperature for 5minutes. Particles were characterized by Nanosight Nanoparticle TrackingAnalysis, Dynamic Light Scattering, and/or Transmission ElectronMicroscopy.

PLGA and PLGA-PEG Particle Characterization:

For sizing, microparticles or nanoparticles were first diluted to 1mg/mL in water or PBS and then sized by DLS, NTA, SEM, or TEM usingappropriate. For loading, particles were first dissolved in DMSO. Forpart of sample, the DMSO sample was added to a larger aqueous volume toprecipitate the PLGA. For labeled peptide, a Biotek Synergy 2 platereader was used to measure fluorescence of the samples and of a labeledpeptide only standard. For non-labeled 2043 samples, PAGE and silverstaining were used to quantify peptide.

SEM Imaging of Microparticles and ImageJ Quantification:

Lyophilized particles were placed on carbon tape (Electron MicroscopySciences, Hatfield, Pa.) and placed on aluminum mounts. Samples weresputtered with gold-palladium, and SEM imaging was performed with aLEO/Zeiss FESEM (Johns Hopkins School of Medicine).

Example 2 SP2043 Peptide (SEQ ID NO:1)

The SP2043 peptide (SEQ ID NO:1) was found to inhibit the proliferation,migration, and adhesion of human umbilical vein endothelial cells(HUVEC; FIG. 1) and of retinal endothelial cells (HREC; FIG. 2). Inaddition, the SP2043 peptide (SEQ ID NO:1) (25 μM) was found to inhibitHUVEC tube formation (FIG. 3, Panels A and B) and microvascularendothelial cell (MEC) tube formation (FIG. 3, Panels C and D). Further,the SP2043 peptide (SEQ ID NO:1) was found to inhibit lymphaticendothelial cell adhesion (LEC; FIG. 4) and LEC tube formation (FIG. 5).

The SP2043 peptide (SEQ ID NO:1) also was found to inhibit hepatocytegrowth factor (HGF) and insulin growth factor 1 (IGF1) signaling invitro in MEC and LEC cells (FIG. 6). In addition, the SP2043 peptide(SEQ ID NO:1) was found to inhibit the proliferation of triple-negativebreast cancer cell lines MDA-MB-231 and SUM149 as well as the estrogenreceptor positive cell line MCF-7 in vitro (FIG. 7). Further, the SP2043peptide (SEQ ID NO:1) was found to inhibit HGF signaling in MDA-MB-231cells (FIG. 8).

The SP2043 peptide (SEQ ID NO:1) also showed inhibition in vivo byinhibiting the growth of MDA-MB-231 orthotopic tumors in SCID mice (FIG.9). 33 days after the injection of the SP2043 peptide (SEQ ID NO:1) intothe mice, the growth of the tumors was inhibited about 82% (using 20 mgof SP2043/kg with an intraperitoneal injection). The SP2043 peptide (SEQID NO:1) also was found to inhibit phosphorylation of c-Met andangiogenesis in vivo (FIG. 10). The SP2043 peptide (SEQ ID NO:1)inhibited angiogenesis as seen by lectin staining forimmunohistochemistry (IHC) and lymphangiogenesis as seen by LYVE-1staining in vivo (FIG. 11). SP2043 inhibited metastasis ofMDA-MB-231-luc tumors to multiple organs in the tumor-conditioned mediapre-treated metastasis model by photon flux from tumor cells (FIG. 12A).The SP2043 peptide (SEQ ID NO:1) also inhibited metastasis ofMDA-MB-231-luc tumors to lymph nodes as seen by staining with vimentinantibody for the presence of human cells (FIG. 12B).

The effect of the SP2043 peptide (SEQ ID NO:1) was also seen in ocularmodels of human ocular diseases such as age-related macular degeneration(AMD), macular edema (ME) and diabetic macular edema (DME). The SP2043peptide (SEQ ID NO:1) inhibited neovascularization in a laser inducedchoroidal neovascularization (CNV) model more potently than aflibercept(FIGS. 13A-B), as well as in a rho-VEGF mouse model compared to acontrol (FIGS. 13C-E). In addition, the SP2043 peptide (SEQ ID NO:1)caused regression of the neovasculature in a laser induced CNV model inthe mouse eye (FIG. 14). Further, the SP2043 peptide (SEQ ID NO:1)inhibited vascular leakage in the Tet/Opsin/VEGF mouse model (FIGS.15A-B). Also, the SP2043 peptide (SEQ ID NO:1) inhibited VEGF mediatedvascular permeability in the rabbit eye (FIG. 16).

Example 3 SP2043 Peptide (SEQ ID NO:1) in Microparticles

The presently disclosed isolated peptides can be formulated and used inmicroparticles or nanoparticles. The SP2043 peptide (SEQ ID NO:1) wasformulated into PLGA microparticles to enable long-term sustainedrelease and shown by scanning electron microscope (SEM) imaging (2%loaded; FIG. 17). FIG. 18A shows a representative example of the sizingof 2% and 5% SP2043 loaded PLGA microparticles. The sizing of themicroparticles was measured with ImageJ (Rasband, 1997-2012) of SEMimages. Results showed that loading with the SP2043 peptide (SEQ IDNO:1) does not significantly affect the particle size distribution. Insome embodiments, about 2% to 5% peptide loaded PLGA is used in thepresently disclosed methods. For in vivo use, 5% by mass SP2043 peptide(SEQ ID NO:1) is equivalent to 1 μg SP2043 peptide (SEQ ID NO:1) in 1 μLof injected microparticle solution. The zeta potentials (surface charge)of 2% and 5% SP2043 peptide (SEQ ID NO:1) loaded microparticles weremeasured on a Malvern Zetasizer (Malvern Instruments, Ltd, Malvern,Worcestershire, UK; FIG. 18B). The results showed no significantdifference in surface charge between the control (blank) and the peptideloaded microparticles.

Controlled release of a labeled peptide analog of SP2043 peptide (SEQ IDNO:1) from PLGA microparticles was observed over six months underphysiological conditions in situ (PBS at 37° C.; FIG. 19A and FIG. 19B).In addition, the controlled release of the SP2043 peptide (SEQ ID NO:1)without any label or modification in PLGA microparticles in situ underphysiological conditions also was observed (FIG. 20).

FIG. 21 shows SEM imaging of PLGA microparticles incorporating 2% byweight of SP2043 peptide (SEQ ID NO:1) (left) and 5% by weight 2043(right). The SP2043 peptide (SEQ ID NO:1) can also be encapsulated intonanoparticles and these particles (micro or nano) can have differentnon-spherical shapes, such as ellipsoidal and spherical shapes (FIG.22).

The PLGA 85/15 microparticle encapsulating the SP2043 peptide (SEQ IDNO:1) caused regression in mouse eyes following laser-induced choroidalneovascularization in a mouse model (FIG. 23). In addition, thesemicroparticles inhibited neovascularization overtime in a laser-inducedwet AMD mouse model (FIG. 24). PLGA 85/15 microparticles encapsulating2043 showed efficacy for at least 3 month in vivo following a singleintravitreal injection.

The PLGA 65/35 microparticles containing the SP2043 peptide (SEQ IDNO:1) inhibited neovascularization overtime in a laser-induced wet AMDmouse model (FIG. 25). Inhibition of neovascularization by the SP2043peptide (SEQ ID NO:1) was also seen in a rabbit model (FIG. 26).

FIG. 27 shows PLGA (85/15) encapsulating the SP2043 peptide (SEQ IDNO:1) in rho/VEGF transgenic mice. FIG. 28 shows that the SP2043 peptide(SEQ ID NO:1) can be used in combination with other anti-angiogenesisagents, such as aflibercept, for increased effect as seen in alaser-induced choroidal neovascularization model in mice.

Polymers, such as PBAEs, can be added to SP2043 to self-assemble it intoapproximately 100 nm nanoparticles (FIG. 29). Particle size andnanoparticle concentration were found to be larger when the SP2043peptide (SEQ ID NO:1) was together with a polymer for self-assembly.FIG. 30 shows the effect of PLGA and PLGA-PEG nanoparticlesencapsulating the SP2043 peptide (SEQ ID NO:1).

Systemically intervenous injected SP2043 peptide (SEQ ID NO:1)containing nanoparticles or free peptide accumulated in tumor as well asother organs (FIG. 31).

The method for quantifying the SP2043 peptide (SEQ ID NO:1) comprisedelectrophoresis and staining with SimplyBlue (FIG. 32, Panel A),followed by mass spectrometry (FIG. 32, Panel B).

Example 4 Discussion

The presently disclosed subject matter provides mimetic peptides thatcan be used for different forms of cancer, ocular diseases, such asage-related macular degeneration, and other angiogenesis-dependent andlymphangiogenesis-dependent diseases. In particular, the active mimeticpeptide SP2043 (SEQ ID NO: 1) was tested in angiogenesis andlymphangiogenesis assays in vitro. More particularly, the SP2043 peptide(SEQ ID NO:1) demonstrated anti-angiogenic activity in blood endothelialcell proliferation, migration, adhesion, and tube formation assays, andanti-angiogenic and anti-tumorigenic activity in vivo in breast cancerxenograft models, and age-related macular degeneration models. TheSP2043 peptide (SEQ ID NO:1) also has anti-lymphangiogenic properties.In addition, it was shown that the SP2043 peptide (SEQ ID NO:1) can beformulated and used in microparticles or nanoparticles.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

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Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method for treating an ocular diseasecomprising administering to a patient an effective amount of a peptidecomprising the amino acid sequence of LRRFSTXPXXXXDINDVXNF (SEQ ID NO:2), wherein X is any amino acid and the amino acid sequence is at least80% identical to LRRFSTAPFAFIDINDVINF (SEQ ID NO: 1), and wherein X atposition 7 is selected from M, A, and G: X at position 9 is selectedfrom F, A, Y, and G: X at position 10 is selected from M, A, G, dA, andNle; X at position 11 is selected from F, A, Y, G, and 4-ClPhe; and X atposition 12 and position 18 are selected from Abu, G, S, A, V, T, I, L,and AllvGly.
 2. The method of claim 1, wherein the ocular disease isselected from age-related macular degeneration (AMD), diabetic macularedema (DME), diabetic retinopathy, macular edema (ME), neovascularglaucoma, and retinopathy of prematurity.
 3. The method of claim 2,wherein the ocular disease is AMD.
 4. The method of claim 3, wherein theAMD is wet AMD.
 5. The method of claim 2, wherein the ocular disease isDME.
 6. The method of claim 2, wherein the ocular disease is diabeticretinopathy.
 7. The method of claim 2, wherein the ocular disease is ME.8. The method of claim 2, wherein the ocular disease is neovascularglaucoma.
 9. The method of claim 2, wherein the ocular disease isretinopathy of prematurity.
 10. The method of claim 1, wherein the aminoacid sequence is at least 85% identical to SEQ ID NO:
 1. 11. The methodof claim 1, wherein the amino acid sequence is at least 90% identical toSEQ ID NO:
 1. 12. The method of claim 1, wherein the amino acid sequenceis at least 95% identical to SEQ ID NO:
 1. 13. The method of claim 1,wherein at least one X is a natural amino acid.
 14. The method of claim1, wherein at least one X is a non-natural amino acid.
 15. The peptideof claim 14, wherein the non-natural amino acid is selected from2-aminobutyric acid (Abu), norleucine (Nle), 4-chloro phenylalanine(4-ClPhe), and allylglycine (AllyGly).
 16. The peptide of claim 1,wherein X at position 7 is A.
 17. The peptide of claim 1, wherein X atposition 9 is F.
 18. The peptide of claim 1, wherein X at position 10 isA.
 19. The peptide of claim 1, wherein X at position 11 is F.
 20. Thepeptide of claim 1, wherein X at position 7 is A, X at position 9 is F,X at position 10 is A, and X at position 11 is F.
 21. The method ofclaim 1, wherein the peptide exhibits anti-angiogenic, anti-vascularpermeability, anti-tumorigenesis, and/or anti-lymphangiogenicproperties.